This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The seven botulinum neurotoxins (BoNTs) originate from the Clostridium botulinum species of bacteria and are, without exception, the most potent toxins known to man- bypassing tetanus, cobra, and ricin toxins in their lethal nature. The contexts in which humans may encounter toxins are many and include bioterrorist attacks, cosmetic and non-cosmetic medical applications, and food poisoning. Indeed, the B serotype (BoNT/B), while not at the forefront of the scientific community's attention heretofore, has been shown to be responsible for a significant portion of annual food poisoning cases caused by this family of toxins. Given the similarities in protein structure between BoNT serotypes and their common catalytic mechanisms, it is not unreasonable to suppose that BoNT/B may also be used as part of a large-scale attack. Ultimately, the goal of our studies is to synthesize efficacious BoNT/B inhibitors that are as exquisitely specific to the toxin as possible so as to avoid nonspecific interactions with other macromolecules in the body and hence unanticipated side effects. To this end, we hope to gain knowledge of the biophysical interactions between ligands pre-screened for optimal activity and the active site of the BoNT/B light chain (BLC), the catalytic subunit of the holotoxin. Applying x-ray diffraction and molecular modeling tools to a truncated form of the BLC (tBLC) will attain this;the full-length construct has proven difficult to crystallize. While we have attained the first tBLC crystals, refinement of the crystallization conditions is required to improve their quality and is something we deem feasible by mid April.Additionally, we would ideally like to optimize existing candidate scaffold(s) and/or develop them de novo. To achieve this goal, the Multiple Solvent Crystal Structures (MSCS) empirical algorithm will be employed to identify exo-sites on the protein surface by examining the localization of organic solvents with distinct chemical functionalities in chemically cross linked crystals. This will help improve specificity of inhibitor binding by exploring binding sites external to the catalytic site that may be specific for chemical groups with defined electrostatic or steric properties. This approach, depending on how tBLC crystallization refinement progresses, might also be possible to pursue during RapiData.